Method for Identifying an inhibitor of plasmidic classic C beta-lactamase having extended-substrate specifity

ABSTRACT

The present invention related to a method for crystallizing a CMY-10 being a β-lactamase with extended-substrate spectrum, a crystal of CMY-10, and a crystal structure of CMY-10. With utilization of three-dimensional structure of CMY-10 protein provided by the present invention, it is possible to develop novel antibiotics or inhibitors that can prevent an emergence of resistance bacteria appeared by plasmidic class C β-lactamases having extended-substrate specificity.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 11/659,244 which is a U.S. national phase application, pursuant to35 U.S.C. §371, of PCT international application Ser. No.PCT/KR2005/002400, filed Jul. 25, 2005, which claims priority to KoreanApplication No. 10-2004-0058283 filed on Jul. 26, 2004 and KoreanApplication No. 10-2005-0041342 filed on May 17, 2005. The entirecontents of the aforementioned patent applications are incorporatedherein by this reference.

TECHNICAL FIELD

The present invention relates to a crystal structure of CMY-10 fromEnterobacter aerogenes, a β-lactamase of plasmid class C causingresistance for β-lactam antibiotics, more precisely to a method forcrystallizing a CMY-10 being a β-lactamase with extended-substratespectrum, a crystal of CMY-10, and a crystal structure of CMY-10.

BACKGROUND ART

β-Lactam antibiotic, including penicillins, cephalosporins, monobactams,and carbapenems induces a death of live cell by inhibition of cell-wallsynthesis (Tomasz, 1979). But it is induced an emergence of bacteriahaving a resistance for the above β-lactam antibiotics due to a broaduse of these antibiotics. Expression of β-lactamase is a generalresistance mechanism of bacteria for β-lactam antibiotics, which theseenzymes hydrolyze a lactam-ring of the above antibiotics. β-Lactamase isclassified into four classes of A, B, C, and D according to homology ofamino acid sequence (Ambler, 1980).

The β-lactamase-mediated resistance of pathogenic bacteria toantibiotics is a continuing threat to public health. Therefore, a thirdgeneration of cephalosporins was developed that could escapeinactivation by β-lactamases. The new antibiotics such as cefotaxime andceftazidime contain bulky oxyimino group at the C7 position ofcephalosporin nucleus. After clinical use, however, novel β-lactamasesthat could inactivate even the oxyimino 1-lactams appeared. For example,the chromosomal class C β-lactamase that hydrolyzes the above oxyiminoβ-lactams has been isolated from the Gram-negative bacteria,Enterobacter cloacae strain GC1 (Nukaga et al., 1995).

Clinically, class A and C β-lactamase are the most commonly encounteredof the four classes. However, class C β-lactamases are more problematicthan class A enzymes. Class C β-lactamases can confer resistance tocephamycins (cefoxitin and cefotetan), penicillins, cephalosporins andβ-lactam/β-lactamase inhibitor combinations and are not significantlyinhibited by clinically used β-lactamase inhibitor such as clavulanicacid. In contrast, Class A β-lactamases are not able to conferresistance to cephamycins and the enzymes are generally susceptible toinhibition by clavulanic acid.

Class C β-lactamases are typically synthesized by the Gram-negativebacteria and are mainly chromosomal. Recently, plasmid-encoded class Cβ-lactamases have been reported in several bacteria species (Lee et al.,2002). Plasmid-encoded class C β-lactamases pose more problems sincethey are transmissible to other bacterial species and are oftenexpressed in a large amount (Marchese et al., 1998).

CMY-1 is the first plasmidic class C β-lactamase to be identified.CMY-10 is a variant of the above CMY-1 with a point mutation at position346 from Asn to Ile. CMY-1 and CMY-10 display the characteristics ofextended-spectrum β-lactamase (ESBLs) (Lee et al., 2003; Horii et al.,1993). The above CMY-10 enzyme is able to hydrolyze cefoxitin andcefotetan as well as penicillins, the third-generation cephalosporins,and monobactams (Lee et al., 2003; Bauernfeind et al., 1989). The highsequence identity between plasmidic β-lactamase and chromosomallactamase clearly defines the origin of the above plasmidic enzymes.Namely, MIR-1, plasmidic β-lactamase, shows over 90% sequence identityto a chromosomal enzyme AmpC from Enterobacter cloacae. P99 is not ESBL,but is wild type of GC1. In the case of CMY-1 and CMY-10, however, theroot is obscure since there is no closely related chromosomal class Cenzyme.

Structural information on class C β-lactamase is very restricted. Allavailable structures have been determined using chromosomal β-lactamase(Crichlow et al., 1999; Lobkovsky et al., 1993; Oefner et al., 1990;Usher et al., 1998). Thus, the structure of CMY-1 and CMY-10 will opennew opportunities for structural comparison between chromosomal andplasmidic class C β-lactamases and for the design of new antibioticsthat can escape hydrolysis by plasmidic class C ESBLs.

With considering the above described state, therefore, it is found theCMY-10 gene present in the plasmid isolated from Enterobacter aerogeneshas been over-expressed in E. coli, followed purification andcrystallization of CMY-10 according to the present invention. It is alsoobtained X-ray diffraction data for CMY-10 crystal and determined athree-dimensional structure of the CMY-10 molecule using the above data.

SUMMARY OF DISCLOSURE

The present invention is to allow using CMY-10, plasmidic class Cβ-lactamase, to develop new antibiotics that can prevent resistantbacteria from emerging by escaping hydrolysis by the above β-lactamasewith determination of a three-dimensional structure of CMY-10 moleculefrom Enterobacter aerogenes.

To achieve the above objection, the present invention provides a methodfor crystallizing a CMY-10 being a class C β-lactamase withextended-substrate spectrum, a crystal of CMY-10, and a crystalstructure of CMY-10.

The present invention will be explained in details hereinafter.

First, a method for crystallizing a CMY-10 is provided. This method ischaracterized in that it consists of (1) a step providing a purifiedCMY-10 protein; (2) a step crystallizing a purified CMY-10 protein withMicrobatch crystallization method at 298K setting using precipitatingagent containing 18% (w/v) polyethylene glycol 8000, 0.1 M sodiumcacodylate (pH6.5) and 0.2 M zinc acetate dehydrate (condition No. 45 ofCrystal Screen™ from Hampton Research); and (3) an analyzing step usinga X-ray crystallography to obtain a three-dimensional structure of theabove crystallized CMY-10 protein.

At this time, in the above step (1), it is used as PCR template aplasmid pYMG-1 containing bla_(CMY-10) gene encoding β-lactamaseproduced by Enterobacter aerogenes which shows resistance topenicillins, three-generation cephalosporins, and monobactams as well ascefoxitin and cefotetan and is isolated at Kosin university gospelhospital, Korea in 1998. The used primer is N-NdeIF and C-XhoIB. Theabove two primers contain recognition sequences for NdeI (N-NdeIF) andXhoI (C-XhoIB) at each end. A method of Lee et al. is used for PCRamplification using DNA thermal cycler (mod 2400; Perkin-Elmer Cetus,Norwalk, Conn., USA). PCR product of desired size 1179 by is confirmedthrough an agarose gel electrophoresis. PCR product digested byNdeI/XhoI is ligated at pET-26b(+) vector (Novagen, Wisconsin, WI, USA)digested with NdeI/XhoI. The hybrid plasmid is designated as pYMG1001.To amplify signal peptide (SP) portion fused with promoter site ofpYMG1001 and His₁₁ (11-histidine), pYMG1001 is used as PCR template, andUP-26b-BglII and Sp-HIS as primer, thereby PCR product of HIS-CMY-SPbeing obtained. To amplify CMY-10 gene fused with enterokinaserecognition site, pYMG-1 is used as PCR template and, EK-CMY and theabove C-XhoIB as primer, thereby PCR product of EK-CMY being obtained.Ligation between blunt ends of the above PCR products, HIS-CMY-SP andEK-CMY, is carried out. Ligation product digested with BglII/XhoI isligated at pET-26b(+) vector (Novagen, Wisconsin, WI, USA) digested withBglII/XhoI. In order to over-express the above His₁₁-bla_(CMY-10), theabove recombinant plasmid DNA(pET-26b/His₁₁-bla_(CMY-10)) is transformedto Escherichia coli strain BL21(DE3), andIPTG(isopropyl-1-thio-β-galactopyranoside) is added to induce anexpression with a large amount of CMY-10 in culture broth of the abovetransformed cell, followed centrifugation of the above cell andre-suspension using 20 mM sodium phosphate buffer (pH 7.0, ice-coldphase). DNase I (100 ug/mL) and 1 mM PMSF (phenylmethyl sulfonylfluoride) is added to the above suspension solution. After lysis of theabove cell, a crude lysate is centrifuged again, a clarified supernatantis loaded His-Bind column (Novagen, Wisconsin, WI, USA) equilibratedwith a binding buffer (20 mM sodium phosphate, 10 mM imidazole, and 500mM NaCl pH 7.9). And then, His₁₁ tag is removed from His₁₁-CMY-10 byenterokinase. The resulting product is desalted and concentrated withFast Desalting column (Amersham Biosciences, UK) and then loaded to MonoS column (Amersham Biosciences, UK) equilibrated previously with 10 mMsodium phosphate buffer (pH 7.0). The nucleotide sequence of the CMY-10isolated and purified by the above procedure is presented as SEQ ID NO:1, and registered at GenBank with registration No. AF357598.

Next, a crystallization of CMY-10 protein in the above step (2) can becarried out by the Microbatch crystallization method at 298K set up orthe hanging-drop vapor-diffusing method at a plate for culturing 24-welltissue (Supercon, South Korea), and it is obtained with a crystal of 0.3mm size using a precipitating agent containing 18% (w/v) polyethyleneglycol 8000, 0.1 M sodium cacodylate (pH 6.5), and 0.2 M zinc acetatedehydrate (condition No. 45 of Crystal Screen™ from Hampton Research).Lastly, a step analyzing a three-dimensional structure of CMY-10 proteincrystal using an X-ray crystallography in the above step (3) can becarried out as following. Namely, it can be determined by obtaining anX-ray diffraction data from a cold substance of CMY-10 protein crystal,calculating an electric density from the above data and using awell-known computer program for modeling the protein. In the presentinvention, it is processed with a CNS program base on an X-raydiffraction data collected at 1.55 Å resolution.

The present invention provides a crystal of CMY-10 protein crystallizedaccording to the above method.

The amino acid sequence of CMY-10 crystal according to the presentinvention is presented as SEQ ID NO: 2, and its space group is P2₁. Thecrystal of CMY-10 protein has unit cell parameters, a=49.70, b=59.51,c=63.75 Å and β=102.57°, and one CMY-10 molecule is included inasymmetric unit of the crystal.

The present invention also provides three-dimensional structure ofCMY-10 protein crystallized according to the above method.

A ribbon diagram of three-dimensional structure of CMY-10 protein isconsisted of α-domain and α/β-domain. The α-domain has three α-helicesand loops. The α/β domain folds as an eight stranded antiparallelβ-sheet with eight α-helices and three β-strands (β3, β4, and β7) packedon both faces of the sheet, and with two β-strands (β5 and β6) on oneedge (α-helix: 11; β-strand: 13). An active site is located at a center,the upper active site is R1 site and the lower active site is R2 site.The R1 site is a space formed by flexible part of Ω loop positioned atthe upper left side, Gln121 loop positioned at the most left side amongα-domain and defining the edges of the active site, and β11 strandpositioned at the most right side among β/β-domain and defining theedges of the active site. The R2 site is a space formed by α10, α11helix, and Tyr151 loop positioned at the lower site. Unlike CMY-10, P99has not extended-substrate spectrum due to its characteristic which isunable to hydrolyze third-generation cephalosporins. With comparingC_(α) backbone diagram of P99 (representing a 40.16% sequence identityto CMY-10) with C_(α) backbone diagram of CMY-10, the distance betweenα10 and adjacent α11 of R2 is extended to 2 Å due to deletion of aminoacid sequence (PPA) presenting at from 303 position to 305 position inP99, and the distance between Gln121 loop and β11 strand of R1 isextended to 1 Å because residues 83-106 in the helical domain formsolvent exposed loops that display the large structural deviation. Thesequence difference between CMY-10 and P99 β-lactamase in this loopregion is 68%. The extension of the above active site, R1 and R2 site,allows to bind a bulky oxyimino group presented at C7 position ofnucleus of ceftazidime (a third-generation cephalosporin) at an activesite of CMY-10, thereby CMY-10 hydrolyzing ceftazidime. With viewing asurface diagram which is a real feature of CMY-10, CMY-10 showsextended-substrate spectrum by binding with third-generationcephalosporins (ceftazidime) due to the above two extended sites able toreceive oxyimino group of ceftazidime, and this phenomenon is newcharacteristic that only CMY-10 has. The three-dimensional structuralatomic coordinates of the above CMY-10 is deposited to Protein Data Bank(PDB) with deposition No. 1ZKJ on May 3, 2005.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

Other objects and aspects of the present invention will become apparentfrom the following description of embodiments with reference to theaccompanying drawing in which:

FIG. 1 is a photograph of CMY-10 crystal from Enterobacter aerogenes,which is crystallized according to the present invention.

FIG. 2 shows three-dimensional molecular structure of CMY-10 fromEnterobacter aerogenes by X-ray crystallography according to the presentinvention with ribbon diagram, the right of the above diagram isα-domain of CMY-10, the left is α/β-domain, and the central pouch shapeis an active site, respectively. α-Helix is represented with blue color,and β-sheet is represented with yellow color.

FIG. 3 is C_(α) backbone diagram showing a position of α-domain (theright), α/β-domain (the left), and an active site (the central pouchshape) of CMY-10 from Enterobacter aerogenes according to the presentinvention.

FIG. 4 is C_(α) backbone diagram showing a position of α-domain (theright), α/β-domain (the left), and an active site (the central pouchshape) of CMY-10 (yellow) from Enterobacter aerogenes and P99 (Red; PDBfile 1BLS; representing a 40.16% sequence identity to CMY-10) fromEnterobacter cloacae according to the present invention.

FIG. 5 is C, backbone diagram of CMY-10 (yellow) and P99 (Red; PDB file1BLS; representing a 40.16% sequence identity to CMY-10), showing thefirst widening position (being indicated with lower white circle, R2active site being extended to 2 Å) of R2 active site (the central pouchshape) of CMY-10 from Enterobacter aerogenes by deletion of amino acidsequence (PPA) being presented at position 303 to position 305 in P99according to the present invention.

FIG. 6 is C_(α) backbone diagram of CMY-10 (yellow) and P99 (Red; PDBfile 1BLS; representing a 40.16% sequence identity to CMY-10), showingthe second widening position (being indicated with upper white circle,R1 active site being extended to 1 Å) of R1 active site (the centralpouch shape) of CMY-10 from Enterobacter aerogenes by difference ofamino acid residues from 83 to 106 between CMY-10 and P99 according tothe present invention.

FIG. 7 is surface diagram showing a position of α-domain, α/β-domain,and an active site (the central pouch shape) of CMY-10 from Enterobacteraerogenes according to the present invention.

FIG. 8 is surface diagram of CMY-10 from Enterobacter aerogenesaccording to the present invention, showing extended-substrate spectrumby binding with third-generation cephalosporins (ceftazidime; centralcompound) due to two extended sites able to receive oxyimino group ofceftazidime and shown at FIG. 6 and FIG. 7.

DETAILED DESCRIPTION OF DISCLOSURE

A greater understanding of the present invention and its concomitantadvantages will be obtained by referring to the following Example andComparative example provided, but it is not to limit the scope of thepresent invention.

Example 1 Overexpression and Purification of CMY-10

To express CMY-10 as a histidine-tagged fusion form, the plasmid pYMG-1(Lee et al., 2003) is used as PCR template and the plasmid containsbla_(CMY-10) gene encoding β-lactamase produced by Enterobacteraerogenes which shows resistance to penicillins, three-generationcephalosporins, and monobactams as well as cefoxitin and cefotetan andis isolated at Kosin university gospel hospital, Korea in 1998. The usedprimer is N-NdeIF (5′-GTAGACCATATGCAACAACGACAATCCATCCTGTGG-3′ (SEQ IDNO: 3), containing NdeI recognition site shown in a bold-type) andC-XhoIB (5′-GAATGTCTCGAGCTCTTTCTTTCAACCGGCCAAC-3′ (SEQ ID NO: 4),containing XhoI recognition site shown in a bold-type). Two primerscontain recognition sequences (bold-type) for NdeI (N-NdeIF) and XhoI(C-XhoIB) at each end. A method of Lee et al. is used for PCRamplification using DNA thermal cycler (mod 2400; Perkin-Elmer Cetus,Norwalk, CT, USA). PCR product of desired size 1179 by is confirmedthrough an agarose gel electrophoresis. PCR product digested byNdeI/XhoI is ligated at pET-26b(+) vector (Novagen, Wisconsin, WI, USA)digested with NdeI/XhoI. The formed plasmid is named as pYMG1001. Toamplify signal peptide (SP) portion fused with promoter site of pYMG1001and His₁₁ (11-histidine), pYMG1001 is used as PCR template, andUP-26b-BglII (5′-CTATCATGCCATACCCGCAAAG-3′ (SEQ ID NO: 5), containingBglII recognition site derived from pET26b(+) in PCR product) and Sp-HIS(5′-GCTATGATGATGATGATGATGATGATGATGATGATGATCCGGTGAAGCCTCACCTGCAT G-3′(SEQ ID NO: 6), containing His₁₁ site shown in a bold-type) as primer,thereby PCR product of HIS-CMY-SP being obtained. To amplify CMY-10 genefused with enterokinase recognition site, pYMG-1 is used as PCR templateand, EK-CMY (5T-AGCGGCCATATCGACGACGACGACAAGGGTGAGGCTICACCGGICGATC-3′(SEQ ID NO: 7), containing enterokinase recognition site shown in abold-type) and the above C-XhoIB as primer, thereby PCR product ofEK-CMY being obtained. Ligation between blunt ends of the above PCRproducts, HIS-CMY-SP and EK-CMY, is carried out. Ligation productdigested with BglII/XhoI is ligated at pET-26b(+) vector (Novagen,Wisconsin, WI, USA) digested with BglII/XhoI. The formed plasmid isnamed as pET-26b/His_(n)-bla_(CMY-10). After verifying the above DNAsequence, in order to over-express the above His₁₁-bla_(CMY-10), theabove recombinant plasmid DNA is transformed into Escherichia colistrain BL21(DE3). The transformed cells are grown in Luria-Bertanimedium (Difco) containing 50 ug/mL of kanamycin to an OD₆₀₀ of 0.6 at303K and expression of CMY-10 is induced with 0.5 mM IPTG(isopropyl-1-thio-β-galactopyranoside) for 16 h at 301K. Cells areharvested by centrifugation at 5000 g for 10 min at 227K andre-suspended in ice-cold 20 mM sodium phosphate buffer pH 7.0. DNase I(100 ug/mL) and 1 mM PMSF (phenylmethyl sulfonyl fluoride) are added tothe above suspension and cells are disrupted by sonication. The crudelysate is centrifuged at 20,000 g for 30 min at 277K and the clarifiedsupernatant is loaded onto a His-Bind column (Novagen, Wisconsin, WI,USA) equilibrated with binding buffer (20 mM sodium phosphate, 10 mMimidazole, and 500 mM NaCl pH 7.9). For further purification, His₁₁ tagis removed from enterokinase according to the instruction ofmanufacturer, Novagen. The reaction mixture is desalted and concentratedwith Fast Desalting column (Amersham Biosciences, UK) and then loadedonto Mono S column (Amersham Biosciences, UK) pre-equilibrated with 10mM sodium phosphate buffer (pH 7.0). The soluble form of CMY-10 withoutHis₁₁ tag is obtained with a yield of 9.2 mg of homogeneous protein perliter of culture. The purified CMY-10 is dialyzed against 10 mMphosphate buffer and subsequently concentrated to 17 mg/mL forcrystallization. Like other class C β-lactamase, the apparent molecularweight of the purified CMY-10 is estimated to be 38 kDa by SDS-PAGE.

Example 2 Microbatch Crystallization of CMY-10

Crystals of CMY-10 is obtained by the batch-crystallization method at298K set up by using an automatic crystallization machine, IMPAX 1-5system (Douglas Instruments Ltd, UK). 1 uL of protein solution and anequal volume of crystallization regent are pipetted under a layer of a1:1 mixture of silicon oil and paraffin oil in 72-well plate (Nunc).Initial crystallization conditions are tested by using all the availablescreening kits from Hampton Research and Emerald BioStructures Inc. As aresult, the crystal of 0.3 mm size is produced by using a precipitatingagent containing 18% (w/v) polyethylene glycol 8000, 0.1 M sodiumcacodylate (pH 6.5), and 0.2 M zinc acetate dehydrate (condition No. 45of Crystal Screen™ from Hampton Research). The crystal of CMY-10 isshown in FIG. 1.

As a result, the crystal of CMY-10 protein is revealed to be belonged tothe monoclinic space group P2₁ with unit-cell parameters a=49.70,b=59.51, c=63.75 Å and β=102.57°, and the crystal volume per unitmolecular weight (V_(M)) is calculated to be 2.25 Å³ Da⁻¹ with a solventcontent of 44.84% (v/v) when the unit cell is assumed to contain twomolecules. This corresponds to one molecule per asymmetric unit. Thestatistics of data collection is shown in table 1.

Table 1

Characteristics of CMY-10 Crystal and Analysis of Data-CollectionStatistics.

Protein CMY-10 Wavelength (Å) 1.12714 Space group P2₁ Unit-cellparameters (Å, °) a = 49.70, b = 59.51, c = 63.75, β = 102.57 Resolutionrange (Å) 20.0-1.55 Completeness (>0σ) (%) 96.6 (99.6) Total/uniquereflections 267900/50744 R_(sym) ^(†) (%) 5.8 (18.1) I/σ (I) 30.77^(†)R_(sym) = Σ | I_(obs) − I_(avg) | /ΣI_(obs)

Example 3 Three-Dimensional Structure Determination and Refinement ofCMY-10 Protein

To obtain an X-ray data from the crystal of the above CMY-10, thecrystal is soaked in a cryoprotectant solution consisted of aprecipitant solution containing 15% (v/v) glycerol for a while and thenflash-cooled with a nitrogen gas of 100 K by using a cooler (OxfordCryosystems, UK). Diffraction data is collected from the above cooledCMY-10 crystal by using a MacScience 2030b area detector at beamline 6Bof Pohang light Source (6B, PLS), South Korea. At this time, thewavelength of synchrotron radiation is 1.12714 Å. A total of 90 framesof 2° oscillation are measured with the crystal-to-detector distance setto 300 mm.

An X-ray diffraction data is collected at 1.551 resolution and processedwith the program CNS (Otwinowski & Minor, 1997). After the X-ray databeing obtained, an electric density map is calculated from the abovedata and model building of CMY-10 is carried out. In a quality analysisof a model, PROCHECK (Laskowski et al., 1993) program is used, and theresults show that 83.1% among 840 ordered residues are in the mostfavored regions, 15.2% are in additionally allowed regions, 1.1% are ingenerously allowed regions, and only 0.7% are in disallowed regions.FIG. 2 to FIG. 8 are drawn up by using Raster 3D (Merrit and Murphy,1994) and Molscript (Kraulis, 1991) program.

As a result, a ribbon diagram (see FIG. 2) of three-dimensionalstructure of CMY-10 protein (359 amino acid residues) is consisted ofα-domain (residues 83-170) and α/β-domain (residues 1-82 and 171-359).The α-domain has three α-helices and loops. The α/β domain folds as aneight stranded antiparallel β-sheet with eight α-helices and threeβ-strands (β3, β4, and β7) packed on both faces of the sheet, and withtwo β-strands (β5 and β6) on one edge (α-helix: 11; β-strand: 13). Anactive site is located at a center, the upper active site is R1 site andthe lower active site is R2 site. The R1 site is a space formed byflexible part (residues 212-226) of Ω loop positioned at the upper leftside, Gln121 loop (residues 118-128) positioned at the very left sideamong α-domain and defining the edges of the active site, and β11 strandpositioned at the very right side among α/β-domain and defining theedges of the active site. The R2 site is a space formed by α10, α11helix and Tyr151 loop (residues 149-152) positioned at the lower site.Unlike CMY-10, P99 has not extended-substrate spectrum due to itscharacteristic which is unable to hydrolyze third-generationcephalosporins. With comparing C_(α) backbone diagram (see FIG. 4) ofP99 (representing a 40.16% sequence identity to CMY-10) with C_(α)backbone diagram (see FIG. 3 and FIG. 4) of CMY-10, the location andgeometry of catalytic residues such as Ser65, Tyr151, the nucleophileand the main chain nitrogen atoms of Ser65 and Ser315 that form theoxyanion hole, are well conserved in P99 and CMY-10. But, the distancebetween α10 and adjacent α11 of R2 is extended to 2 Å because of thedeletion of amino acid sequence (PPA) presenting from 303 position to305 position in P99 (see white circle at FIG. 5), and the distancebetween Gln121 loop and β11 strand of R1 is extended to 1 Å becauseresidues 83-106 in the helical domain form solvent exposed loops thatdisplay the large structural deviation (see white circle at FIG. 6). Thesequence difference between CMY-10 and P99 β-lactamase in this loopregion is 68%. The extension of the above active site, R1 and R2 site,allows to bind a bulky oxyimino group presented at C7 position ofnucleus of ceftazidime (a third-generation cephalosporin) at an activesite of CMY-10, thereby CMY-10 hydrolyzing ceftazidime. With viewing asurface diagram (see FIG. 7) which is a real feature of CMY-10, CMY-10shows extended-substrate spectrum by binding with third-generationcephalosporins (ceftazidime; central compound in FIG. 8) due to theabove two extended sites able to receive oxyimino group of ceftazidime,and this phenomenon is new characteristic that only CMY-10 has.Extended-substrate specificity of CMY-10 is produced by a new mechanismdifferent from that of a chromosomal β-lactamase from E. cloacae GC1(representing a 39.79% sequence identity to CMY-10) whosethree-dimensional structure is only known among a class C ESBL havingextended-substrate specificity for third-generation cephalosporins. Theinsertion mutation consisting of an unusual tandem repeat of threeresidues (Ala208-Val209-Arg210) in Ω-loop is responsible for theextended activity of the GC1 β-lactamase, which widens the active siteenough to accommodate the oxyimino group of third-generationcephalosporins (Crichlow et al., 1999). However, CMY-10 does not havesuch an insertion mutation, and shows the extension of R1 and R2 activesite and a new three-dimensional structural specificity describing amechanism of extended-substrate spectrum for third-generationcephalosporins. The three-dimensional structural atomic coordinates ofthe above CMY-10 is deposited to PDB with deposition No. 1ZKJ on May 3,2005.

As described in detail through the above example, the present inventionrelates to a method for crystallizing a CMY-10 being a β-lactamase withextended-substrate spectrum, a crystal of CMY-10, and a crystalstructure of CMY-10. With utilization of three-dimensional structure ofCMY-10 protein as the above description, it is possible to develop novelantibiotics or inhibitors being capable of preventing an emergence ofresistance bacteria appeared by plasmidic class C β-lactamases havingextended-substrate specificity, therefore the present invention is veryuseful in medical industry.

1. A method for identifying a potential inhibitor of plasmidic class Cβ-lactamases having extended-substrate specificity, the methodcomprising the steps of: (a) providing a crystal of CMY-10 proteincomprising the amino acid sequence set forth in SEQ ID NO: 2, thecrystal having unit cell parameters of a=49.70 Å, b=59.51 Å, c=63.75 Åand β=102.57° and having its space group as P2₁; (b) determining athree-dimensional structure of said crystal; (c) designing or selectinga potential inhibitor of plasmidic class C β-lactamases by employing thethree-dimensional structure; (d) synthesizing the potential inhibitor;and (e) contacting the potential inhibitor with CMY-10 protein in thepresence of a substrate and measuring the ability of the potentialinhibitor to inhibit CMY-10 protein.